3-iodotyrosine fluorescent proteins for improved expansion microscopy

Expansion microscopy is a powerful technique for achieving nanoscale resolution, but labelling and detection using fluorescent proteins (FPs) have been hampered by signal degradation during expansion. Current approaches therefore rely heavily on immunostaining, ssDNA-FISH or HALO/SNAP-based detection systems. Here we show that substituting tyrosine with 3-iodotyrosine in the chromophore of superfolder GFP dramatically improves signal retention during expansion microscopy in zebrafish embryos. This enhancement specifically protects FPs during the formaldehyde-dependent anchoring step, and may additionally reflect better tolerance of the prolonged acidic conditions encountered during anchoring. 3-iodotyrosine-modified FPs open a door to reliable protein-based expansion microscopy.